Succinic acid derivatives

ABSTRACT

The present invention relates to compounds of the general formula (I), processes for their preparation, pharmaceutical compositions containing them as well as their use for the production of pharmaceutical compositions for the treatment of inflammatory diseases

This application is a 371 of PCT/EP02/06941, filed Jun. 24, 2002.

The present invention relates to compounds of formula (I),

their preparation and use as pharmaceutical compositions as integrinantagonists, especially as α₄β₁ and/or α₄β₇ and/or α₉β₁ integrinantagonists and in particular for the production of pharmaceuticalcompositions suitable for the inhibition or the prevention of celladhesion and cell-adhesion mediated disorders.

Adhesive interactions between the leukocytes and endothelial cells playa critical role in leukocyte trafficking to sites of inflammation. Theseevents are essential for normal host defense against pathogens andrepair of tissue damage, but can also contribute to the pathology of avariety of inflammatory and autoimmune disorders. Indeed, eosinophil andT cell infiltration into the tissue is known as a cardinal feature ofallergic inflammation such as asthma.

The interaction of circulating leukocytes with adhesion molecules on theluminal surface of blood vessels appears to modulate leukocytetransmigration. These vascular cell adhesion molecules arrestcirculating leukocytes, thereby serving as the first step in theirrecruitment to infected or inflamed tissue sites. Subsequently, theleukocytes reaching the extravascular space interact with connectivetissue cells such as fibroblasts as well as extracellular matrixproteins such as fibronectin, laminin, and collagen. Adhesion moleculeson the leukocytes and on the vascular endothelium are hence essential toleukocyte migration and attractive therapeutic targets for interventionin many inflammatory disorders.

Leukocyte recruitment to sites of inflammation occurs in a stepwisefashion beginning with leukocyte tethering to the endothelial cellslining the blood vessels. This is followed by leukocyte rolling,activation, firm adhesion, and transmigration. A number of cell adhesionmolecules involved in those four recruitment steps have been identifiedand characterized to date. Among them, the interaction between vascularcell adhesion molecule 1 (VCAM-1) and very late antigen 4 (VLA-4, α₄β₁integrin), as well as the interaction between mucosal addressin celladhesion molecule 1 (MAdCAM-1) and α₄β₇ integrin, has been shown tomediate the tethering, rolling, and adhesion of lymphocytes andeosinophils, but not neutrophils, to endothelial cells under aphysiologic flow condition. This suggests that the VCAM-1/ VLA-4 and/orMAdCAM-1/α₄β₇ integrin mediated interactions could predominantly mediatea selective recruitment of leukocyte subpopulations in vivo. Theinhibition of this interaction is a point of departure for therapeuticintervention (A. J. Wardlaw, J. Allergy Clin. Immunol. 1999, 104,917–26).

VCAM-1 is a member of immunoglobulin (Ig) superfamily and is one of thekey regulators of leukocyte trafficking to sites of inflammation.VCAM-1, along with intracellular adhesion molecule 1 (ICAM-1) andE-selectin, is expressed on inflamed endothelium activated by suchcytokines as interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α),as well as by lipopolysaccharide (LPS), via nuclear factor κB (NF-κB)dependent pathway. However, these molecules are not expressed on restingendothelium. Cell adhesion mediated by VCAM-1 may be involved innumerous physiological and pathological processes including myogenesis,hematopoiesis, inflammatory reactions, and the development of autoimmunedisorders. Integrins VLA-4 and α₄β₇ both function as leukocyte receptorsfor VCAM-1.

The integrin α₄β₁ is a heterodimeric protein expressed in substantiallevels on all circulating leukocytes except mature neutrophils. Itregulates cell migration into tissues during inflammatory responses andnormal lymphocyte trafficking. VLA-4 binds to different primary sequencedeterminants, such as a QIDSP motif of VCAM-1 and an ILDVP sequence ofthe major cell type-specific adhesion site of the alternatively splicedtype III connecting segment domain (CS-1) of fibronectin.

In vivo studies with neutralizing monoclonal antibodies and inhibitorpeptides have demonstrated a critical role for α₄ integrins interactionin leukocyte-mediated inflammation. Blocking of VLA-4/ligandinteractions, thus, holds promise for therapeutic intervention in avariety of inflammatory, autoimmune and immune diseases (Zimmerman, C.;Exp. Opin. Ther. Patents 1999, 9, 129–133).

Furthermore, compounds containing a bisarylurea moiety as a substituentwere disclosed as α₄β₁ integrin receptor antagonists: WO 96/22966, WO97/03094, W099/20272, W099/26923, WO 99/33789, WO 99/37605, WO 00/00477.However, no β-amino acids or homologues thereof with α₄β₁ integrinreceptor antagonistic activity have been described.

Further to their α₄β₁ integrin antagonistic activity, the compounds ofthe present invention may also be used as α₄β₇ or α₉β₁ integrinantagonists.

An object of the present invention is to provide new succinic acid orhomologues thereof derived integrin antagonists for the treatment ofinflammatory, autoimmune and immune diseases.

The present invention therefore relates to compounds of the generalformula (I)

-   -   wherein    -   R¹ represents a 4- to 9-membered saturated, unsaturated or        aromatic cyclic residue,        -   which can contain 0 to 3 heteroatoms selected independently            from the group N, S and O,        -   and wherein R¹ is substituted by —R¹⁻¹-Z, wherein    -   R¹⁻¹ represents a bond, —O—, —S—, NR¹⁻², C₁–C₁₀ alkyl, C₂–C₁₀        alkenyl, C₂–C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a        4–9-membered saturated or unsaturated heterocyclic residue        containing up to 3 heteroatoms selected from the group oxygen,        nitrogen or sulfur,        -   wherein R¹⁻¹ can optionally be substituted by 1 to 2            substituents selected from the group R¹⁻³,        -   wherein R¹⁻² can optionally be hydrogen, C₁–C₁₀ alkyl,            C₂–C₁₀ alkenyl or C₂–C₁₀ alkynyl, and        -   wherein R¹⁻³ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀            alkenyl, C₂–C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or            a 4–9-membered saturated or unsaturated heterocyclic residue            containing up to 3 heteroatoms selected from the group            oxygen, nitrogen or sulfur,    -   Z represents —C(O)OR^(Z-1), —C(O)NR^(Z-2)R^(Z-3),        SO₂NR^(Z-2)R^(Z-3), —SO(OR^(Z-1)), —SO₂(OR^(Z-1)),        —P(O)R^(Z-1)(OR^(Z-3)) or —PO(OR^(Z-1))(OR^(Z-3)),        -   wherein R^(Z-2) is hydrogen, C₁–C₄ alkyl, C₂–C₆ alkenyl,            C₂–C₆ alkynyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,            —C(O)R^(Z-4) or —SO₂R^(Z-4),        -   wherein R^(Z-4) is C₁–C₄ alkyl, C₂–C₆ alkenyl, C₂–C₆            alkynyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,    -   R^(Z-1) and R^(Z-3) are independently selected from the group        hydrogen, C₁–C₄ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl, C₃–C₆        cycloalkyl, C₆ or C₁₀ aryl or benzyl,        -   wherein R^(Z-1) and R^(Z-3) can optionally be substituted by            1 to 3 substituents selected from the group C₁–C₄ alkyl,            C₁–C₄ alkyloxy, halogen, nitro, cyano,    -   and wherein R¹ can optionally be substituted by 0 to 2        substituents R¹⁻⁴, halogen, nitro, amino, cyano and oxo,    -   wherein    -   R¹⁻⁴ is selected from the group C₁–C₄ alkyl, C₁–C₄ alkyloxy,        phenyl, phenoxy, phenylamino, C₃–C₆ cycloalkyl,    -   R² represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl or C₃–C₇ cycloalkyl,    -   wherein R² can optionally be substituted by 1 to 3 radicals        independently selected from the group C₁–C₄ alkyl,        trifluormethyl, trifluormethoxy, halogen, cyano, nitro or oxo,    -   R³ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered        saturated or unsaturated heterocyclic residue containing up to 2        heteroatoms selected from the group oxygen, nitrogen or sulfur,    -   wherein R³ can optionally be substituted by 1 to 3 radicals        R³⁻¹,    -   wherein R³⁻¹ represents C₁–C₄ alkyl, trifluormethyl,        trifluormethoxy, —OR³⁻², —SR³⁻², NR³⁻³R³⁻⁴, —C(O)R³⁻², S(O)R³⁻²,        —SO₂R³⁻², —OC(O)R³⁻², —C(O)NR³⁻³R³⁻⁴, —NR³⁻²C(O)R³⁻³,        —SO₂NR³⁻³R³⁻⁴, NR³⁻²SO₂R³⁻³, —NR³⁻²C(O)NR³⁻³R³⁻⁴,        —NR³⁻²C(O)OR³⁻³, —OC(O)NR³⁻³R³⁻⁴, —CO₂R³⁻⁵, halogen, cyano,        nitro or oxo,    -   wherein R³⁻² represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl,        C₆ or C₁₀ aryl,    -   wherein R³⁻³ and R³⁻⁴ are independently selected from the group        hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl or        benzyl,    -   and wherein R³⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or        C₁₀ aryl,    -   R⁴ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered        saturated or unsaturated heterocyclic residue containing up to 2        heteroatoms selected from the group oxygen, nitrogen or sulfur,    -   wherein R⁴ can optionally be substituted by 1 to 3 radicals        R⁴⁻¹,    -   wherein R⁴⁻¹ represents C₁–C₄ alkyl, trifluormethyl,        trifluormethoxy, —OR⁴⁻², —SR⁴⁻², NR⁴⁻³R⁴⁻⁴, —C(O)R⁴⁻², S(O)R⁴⁻²,        —SO₂R⁴⁻², —OC(O)R⁴⁻², —C(O)NR⁴⁻³R⁴⁻⁴, —NR⁴⁻²C(O)R⁴⁻³,        —SO₂NR⁴⁻³R⁴⁻⁴, NR⁴⁻²SO₂R⁴⁻³, —NR⁴⁻²C(O)NR⁴⁻³R⁴⁻⁴,        —NR⁴⁻²C(O)OR⁴⁻³, —OC(O)NR⁴⁻³R⁴⁻⁴, —CO₂R⁴⁻⁵, halogen, cyano,        nitro or oxo,    -   wherein R⁴⁻² represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl,        C₆ or C₁₀ aryl,    -   wherein R⁴⁻³ and R⁴⁻⁴ are independently selected from the group        hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl or        benzyl,    -   and wherein R⁴⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or        C₁₀ aryl or    -   R³ and R⁴ together with the carbon atom to which they are        attached form a 4–7-membered saturated or unsaturated ring        containing up to 2 heteroatoms selected from the group oxygen,        nitrogen or sulfur, which can optionally be substituted by 1 to        2 substituents selected from the group C₁–C₄ alkyl, phenyl,        benzyl, C₃–C₇ cycloalkyl, C₁–C₄ alkyloxy, halogen, nitro, cyano,        oxo and which can be fused with a 3–7 membered homocyclic or        heterocyclic, saturated, unsaturated or aromatic ring,    -   R⁵ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl or C₃–C₇ cycloalkyl,    -   wherein R⁵ can optionally up to threefoldedly be substituted by        C₁–C₄ alkyl, trifluormethyl, trifluormethoxy, halogen, cyano,        nitro or oxo,    -   R⁶ represents phenyl or a 5- to 6-membered aromatic heterocyclic        residue containing up to 3 heteroatoms independently selected        from the group oxygen, nitrogen or sulfur,    -   wherein R⁶ is substituted by —NR⁶⁻²C(O)NR⁶⁻³R⁶⁻⁴ or        —NR⁶⁻²C(S)NR⁶⁻³R⁶⁻⁴ and can furthermore optionally be        substituted by halogen,    -   wherein R⁶⁻² and R⁶⁻³ are independently selected from the group        hydrogen or C₁–C₄ alkyl, or together form a group

-   -   and wherein R⁶⁻⁴ represents phenyl,    -   wherein R⁶⁻⁴ can optionally be substituted by 1–2 substituents        selected from the group C₁–C₄ alkyl, C₁–C₄ alkyloxy, halogen,        nitro, trifluoromethyl, trifluoromethoxy or cyano, or    -   R⁶ represents a group

-   -   wherein R⁶⁻¹ represents a substituent selected from the group        hydrogen, C₁–C₄ alkyl, C₁–C₄ alkyloxy, halogen, nitro,        trifluoromethyl, trifluoro-methoxy or cyano, and    -   wherein R⁶⁻⁵ represents a substituent selected from the group        hydrogen, C₁–C₄ alkyl, C₁–C₄ alkyloxy, halogen, nitro,        trifluoromethyl, trifluoro-methoxy or cyano,    -   R⁷ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered        saturated or unsaturated heterocyclic residue containing up to 2        heteroatoms selected from the group oxygen, nitrogen or sulfur,    -   wherein R⁷ can optionally be substituted by 1 to 3 radicals        R⁷⁻¹,    -   wherein R⁷⁻¹ represents C₁–C₄ alkyl, trifluormethyl,        trifluormethoxy, —OR⁷⁻², —SR⁷⁻², NR⁷⁻³R⁷⁻⁴, —C(O)R⁷⁻², S(O)R⁷⁻²,        —SO₂R⁷⁻², —OC(O)R⁷⁻², —C(O)NR⁷⁻³R⁷⁻⁴, —NR⁷⁻²C(O)R⁷⁻³,        SO₂NR⁷⁻³R⁷⁻⁴, NR⁷⁻²SO_(l R) ⁷⁻³, —NR⁷⁻²C(O)NR⁷⁻³R⁷⁻⁴,        —NR⁷⁻²C(O)OR⁷⁻³, —OC(O)NR⁷⁻³R⁷⁻⁴, —CO₂R⁷⁻⁵, halogen, cyano,        nitro or oxo,    -   wherein R⁷⁻² represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl,        C₆ or C₁₀ aryl,    -   wherein R⁷⁻³ and R⁷⁻⁴ are independently selected from the group        hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl or        benzyl,    -   and wherein R⁷⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or        C₁₀ aryl    -   R⁸ represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀        alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered        saturated or unsaturated heterocyclic residue containing up to 2        heteroatoms selected from the group oxygen, nitrogen or sulfur,    -   wherein R⁸ can optionally be substituted by 1 to 3 radicals        R⁸⁻¹,    -   wherein R⁸⁻¹ represents C₁–C₄ alkyl, trifluormethyl,        trifluormethoxy, —OR⁸⁻², —SR⁸⁻², NR⁸⁻³R⁸⁻⁴, —C(O)R⁸⁻², S(O)R⁸⁻²,        —SO₂R⁸⁻², —OC(O)R⁸⁻², —C(O)NR⁸⁻³R⁸⁻⁴, —NR²C(O)R⁸⁻³,        —SO₈₋₂NR⁸⁻³R⁸⁻⁴, NR⁸⁻²SO₂R⁸⁻³, —NR⁸⁻²C(O)NR⁸⁻³R⁸⁻⁴,        —NR⁸⁻²C(O)OR⁸⁻³, —OC(O)NR⁸⁻³R⁸⁻⁴, —CO₂R⁸⁻⁵, halogen, cyano,        nitro or oxo,    -   wherein R⁸⁻² represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl,        C₆ or C₁₀ aryl,    -   wherein R⁸⁻³ and R⁸⁻⁴ are independently selected from the group        hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl or        benzyl,    -   and wherein R⁸⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or        C₁₀ aryl or    -   R⁷ and R⁸ together form a 4–7-membered saturated or unsaturated        ring containing up to 2 heteroatoms selected from the group        oxygen, nitrogen or sulfur, which can optionally be substituted        by 1 to 2 substituents selected from the group C₁–C₄ alkyl,        phenyl, benzyl, C₃–C₇ cycloalkyl, C₁–C₄ alkyloxy, halogen,        nitro, cyano, oxo and which can be fused with a 3–7 membered        homocyclic or heterocyclic, saturated, unsaturated or aromatic        ring,    -   X represents bond or (—CR^(X-1)R^(X-2)—)_(n),    -   wherein R^(X-1) and R^(X-2) can be independently selected from        the group hydrogen, C₁–C₄ alkyl, C₂–C₄ alkenyl, C₂–C₄ alkynyl,    -   wherein R^(X-1) and R^(X-2) can optionally independently be        substituted by 1 to 2 substituents selected from the group C₁–C₄        alkyl, phenyl, benzyl, C₃–C₇ cycloalkyl, C₁–C₄ alkyloxy,        halogen, nitro, cyano, oxo,    -   and wherein n is an integer 0 or 1,    -   and pharmaceutically acceptable salts thereof.

In the context of the present invention alkyl stands for astraight-chain or branched alkyl residue, such as methyl, ethyl,n-propyl, iso-propyl, n-pentyl. If not stated otherwise, preferred isC₁–C₁₀ alkyl, very preferred is C₁–C₆ alkyl.

Alkenyl and alkinyl stand for straight-chain or branched residuescontaining one or more double or triple bonds, e.g. vinyl, allyl,isopropinyl, ethinyl. If not stated otherwise, preferred is C₁–C₁₀alkenyl or alkinyl, very preferred is C₁–C₆ alkenyl or alkinyl.

Cycloalkyl stands for a cyclic alkyl group such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Preferred ismonocyclic C₃–C₇ cycloalkyl.

Halogen in the context of the present invention stands for fluorine,chlorine, bromine or iodine. If not specified otherwise, chlorine orfluorine are preferred.

A 4- to 9-membered saturated, unsaturated or aromatic cyclic residuestands for a monocyclic system containing 4 to 9 ring atoms andcontaining 0, 1 or more double bonds, which can be attached via a carbonatom or eventually via a heteroatom within the ring, for example phenyl,thiazolyl, pyridyl, cyclopentyl.

Aryl stands for a monocyclic Hueckel-aromatic cyclic system containing 6or 10 ring carbon atoms.

Heteroaryl stands for a monocyclic heteroaromatic system containing 4 to9 ring atoms, which can be attached via a carbon atom or eventually viaa nitrogen atom within the ring, for example, furan-2-yl, furan-3-yl,pyrrol-1-yl, pyrrol-2-yl, pyrrol-3-yl, thienyl, thiazolyl, oxazolyl,imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl or pyridazinyl.

A saturated or unsaturated heterocyclic residue stands for aheterocyclic system containing 4 to 9 ring atoms, which can contain oneor more double bonds and which can be attached via a ring carbon atom oreventually via a nitrogen atom, e.g. tetrahydrofur-2-yl,pyrrolidine-1-yl, piperidine-1-yl, piperidine-2-yl, piperidine-3-yl,piperidine-4-yl, piperazine-1-yl, piperazine-2-yl morpholine-1-yl,1,4-diazepine-1-yl or 1,4-dihydropyridine-1-yl.

If not specified otherwise, in the context of the present inventionheteroatom stands preferably for O, S, N or P.

Compounds according to the invention, wherein one of the substituentsR³, R⁴, R⁷ and R⁸ represents phenyl, which can optionally be substitutedby up to three substituents independently selected from the groupC₁–C₄-alkyl and halogen, and the remaining substituents representhydrogen are such that have one single phenyl group bound to any one ofthe chain carbon atoms to which these substituents are attached.

In a preferred embodiment, the present invention relates to compounds ofgeneral formula (1), wherein R¹ represents a phenyl ring.

In another preferred embodiment, the present invention relates tocompounds of general formula (I), wherein R¹⁻¹ represents a bond and Zrepresents COOR^(Z-1), wherein R^(Z-1) has the meaning indicated above.

In yet another preferred embodiment, the present invention relates tocompounds of general formula (I), wherein R⁶ represents phenyl, which issubstituted by —NHC(O)NHR⁶⁻⁴, wherein R⁶⁻⁴ is substituted with methyl ortrifluoromethoxy.

In yet another preferred embodiment, the present invention relates tocompounds of general formula (I), wherein X represents a methylenegroup.

In yet another preferred embodiment, the present invention relates tocompounds of general formula (I), wherein one of the substituents R³,R⁴, R⁷ and R⁸ represents phenyl, which can optionally be substituted byup to three substituents independently selected from the groupC₁–C₄-alkyl and halogen, and the remaining substituents representhydrogen.

In yet another preferred embodiment, the present invention relates tocompounds of general formula (1), wherein R¹ is a 1,4substituted phenylring.

A preferred process for preparation of compounds of general formula (I)has also been found, which comprises reaction of carboxylic acids ofgeneral formula (I′)

or activated derivatives thereof, wherein

R¹, R², R³, R⁴, R⁷ and R⁸ have the above-mentioned meaning, withcompounds of the general formula (I″)R⁶—X—NR⁵H   (I″),wherein

X, R⁵ and R⁶ have the above-mentioned meaning,

in inert solvents, which will be described in more detail in thedescriptive part of the specification.

Surprisingly, the compounds of the present invention show good integrinantagonistic activity. They are therefore suitable for the treatment ofdiseases, especially as α₄β₁ and/or α₄β₇ and/or α₉β₁ integrinantagonists and in the manufacture of a medicament for the treatment orthe prevention of a condition mediated by integrins and in particularfor the production of pharmaceutical compositions for the inhibition orthe prevention of cell adhesion and cell-adhesion mediated disorders.Examples are the treatment and the prophylaxis of atherosclerosis,asthma, chronic obstructive pulmonary disease (COPD), allergies,diabetes, inflammatory bowel disease, multiple sclerosis, myocardialischemia, rheumatoid arthritis, transplant rejection and otherinflammatory, autoimmune and immune disorders.

The integrin antagonists of the invention are useful not only fortreatment of the physiological conditions discussed above, but are alsouseful in such activities as purification of integrins and testing foractivity.

For the treatment of the above-mentioned diseases, the compoundsaccording to the invention can exhibit non-systemic or systemicactivity, wherein the latter is preferred. To obtain systemic activitythe active compounds can be administered, among other things, orally orparenterally, wherein oral administration is preferred.

For parenteral administration, forms of administration to the mucousmembranes (i.e. buccal, lingual, sublingual, rectal, nasal, pulmonary,conjunctival or intravaginal) or into the interior of the body areparticularly suitable. Administration can be carried out by avoidingabsorption (i.e. intracardiac, intra-arterial, intravenous, intraspinalor intralumbar administration) or by including absorption (i.e.intracutaneous, subcutaneous, percutaneous, intramuscular orintraperitoneal administration).

For the above purpose the active compounds can be administered per se orin administration forms.

Suitable administration forms for oral administration are, inter alia,normal and enteric-coated tablets, capsules, coated tablets, pills,granules, pellets, powders, solid and liquid aerosols, syrups,emulsions, suspensions and solutions. Suitable administration forms forparenteral administration are injection and infusion solutions.

The active compound can be present in the administration forms inconcentrations of from 0.001–100% by weight; preferably theconcentration of the active compound should be 0.5–90% by weight, i.e.quantities which are sufficient to allow the specified range of dosage.

The active compounds can be converted in the known manner into theabove-mentioned administration forms using inert non-toxicpharmaceutically suitable auxiliaries, such as for example excipients,solvents, vehicles, emulsifiers and/or dispersants.

The following auxiliaries can be mentioned as examples: water, solidexcipients such as ground natural or synthetic minerals (e.g. talcum orsilicates), sugar (e.g. lactose), non-toxic organic solvents such asparaffins, vegetable oils (e.g. sesame oil), alcohols (e.g. ethanol,glycerol), glycols (e.g. polyethylene glycol), emulsifying agents,dispersants (e.g. polyvinylpyrrolidone) and lubricants (e.g. magnesiumsulphate).

In the case of oral administration tablets can of course also containadditives such as sodium citrate as well as additives such as starch,gelatin and the like. Flavour enhancers or colorants can also be addedto aqueous preparations for oral administration.

For the obtainment of effective results in the case of parenteraladministration it has generally proven advantageous to administerquantities of about 0.001 to 100 mg/kg, preferably about 0.01 to 1 mg/kgof body weight. In the case of oral administration the quantity is about0.01 to 100 mg/kg, preferably about 0.1 to 10 mg/kg of body weight.

It may nevertheless be necessary to use quantities other than thosementioned above, depending on the body weight concerned, the method ofadministration, the individual response to the active compound, the typeof preparation and the time or interval of administration.

Pharmaceutically acceptable salts of the compounds of the presentinvention that contain an acidic moiety include addition salts formedwith organic or inorganic bases. The salt forming ion derived from suchbases can be metal ions, e.g., aluminum, alkali metal ions, such assodium of potassium, alkaline earth metal ions such as calcium ormagnesium, or an amine salt ion, of which a number are known for thispurpose. Examples include ammonium salts, arylalkylamines such asdibenzylamine and N,N-dibenzylethylenediamine, lower alkylamines such asmethylamine, t-butylamine, procaine, lower alkylpiperidines such asN-ethylpiperidine, cycloalkylamines such as cyclohexylamine ordicyclohexylarnine, 1-adamantylamine, benzathine, or salts derived fromamino acids like arginine, lysine or the like. The physiologicallyacceptable salts such as the sodium or potassium salts and the aminoacid salts can be used medicinally as described below and are preferred.

Pharmaceutically acceptable salts of the compounds of the presentinvention that contain a basic moiety include addition salts formed withorganic or inorganic acids. The salt forming ion derived from such acidscan be halide ions or ions of natural or unnatural carboxylic orsulfonic acids, of which a number are known for this purpose. Examplesinclude chlorides, acetates, trifluoroacetates, tartrates, or saltsderived from amino acids like glycine or the like. The physiologicallyacceptable salts such as the chloride salts, the trifluoroacetic acidsalts and the amino acid salts can be used medicinally as describedbelow and are preferred.

These and other salts which are not necessarily physiologicallyacceptable are useful in isolating or purifying a product acceptable forthe purposes described below.

The compounds according to the invention can exist in differentstereoisomeric forms, which relate to each other in an enantiomeric way(image and mirror image) or in a diastereomeric way (image differentfrom mirror image). The invention relates to the enantiomers and thediastereomers as well as their mixtures. They can be separated accordingto customary methods.

General Compound Synthesis

The synthesis of compounds according to the general formula (I) can beillustrated by the following scheme 1:

By coupling of the carboxylic acid derivatives (III) with the amines(II), the amides (WV) can be obtained. Coupling of the activatescarboxylic acids (IV) with the amnines (V) yields the amides (VI). Theremoval of the protecting group PG affords carboxylic acids of type (I).

In the above scheme the depicted ring in formulas (I), (II), (IV), and(VI) represents a cyclic moiety formed by R¹. Activated carboxylic acidsderivatives of this type are known to the person skilled in the art andare described in detail in standard textbooks such as, for example in(i) Houben-Weyl, Methoden der organischen Chemie [Methods of OrganicChemistry], Georg Thieme Verlag, Stuttgart or (ii) Comprehensive OrganicSynthesis, Ed. B. M. Trost, Pergamon Press, Oxford, 1991. The carboxylicacid is preferably activated as, such as, for example,AG=1-hydroxy-1H-benzotriazol and a coupling agents such as, for example,dicyclohexyl-carbodiimid (DCC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidexHCl (EDCI),2-(7-aza-3-oxido-1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexa-fluorophosphate. Other activated carboxylic acid derivatives suchas, for example symmetric anhydrides, mixed anhydrides, N-carboxyanhydrides, halides, or further activated esters e.g. succinyl orpentafluorophenyl esters may also be employed.

In the above scheme PG stand for a suitable protecting group of thecarboxyl group or COOPG stand for the carboxylic group attached to apolymeric resin suitable for solid phase synthesis. Protecting groups ofthis type are known to the person skilled in the art and are describedin detail in T. W. Greene, P. G. Wuts, Protective Groups in OrganicSynthesis, 3^(rd) ed., John Wiley, New York, 1999. The carboxyl group ispreferably esterified, PG being C₁₋₆-alkyl such as, for example, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl,neopentyl, hexyl, a C₃₋₇-cycloalkyl such as, for example, cyclopropyl,cyclopropylmethyl, cyclobutyl, cyclo-pentyl, cyclohexyl, an aryl suchas, for example, phenyl, benzyl, tolyl or a substituted derivativethereof.

Step A

Formation of the amides (IV) can take place by reacting a carboxylicacid anhydride (II) with the desired amine (III) or an acceptable saltthereof.

For example, amides of type (I) can be prepared as follows:

Anhydride Procedure

A solution of carboxylic acid anhydride in an inert solvent is stirredat r.t. After addition of the amine and a non-nucleophilic base such asethyldiisopropylamine or potassium carbonate stirring is continued atr.t. or elevated temperature. After evaporation, the residue wasredissolved in ethyl acetate, washed with aqueous acid and base, driedand evaporated. If necessary the product was purified by trituration orby flash-chromatography or used without further purification.

Compounds of general formula (II) are commercially available, known orcan be prepared by customary methods starting from commerciallyavailable precursors.

Compounds of general formula (IEI) are commercially available, known orcan be prepared by customary methods starting from known carboxylic acidderivatives.

Step B

Formation of the amides (VI) can take place by reacting the respectivecarboxylic acids (IV)—activated by a coupling agent such as DCC andHOBt; EDCI and HOBt or HATU—with the desired amines (V) or an acceptablesalt thereof. Activated derivatives of the acids (IV) such asanhydrides, halides, and esters e.g. succinyl or pentafluorophenylesters may also be employed.

For example, amides (VI) can be prepared as follows:

A solution of carboxylic acid, 1-hydroxy-1H-benzotriazol (HOBt) and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidexHCl (EDCI) in an inertsolvent is stirred at r.t. After addition of the amine and anon-nucleophilic base such as ethyldiisopropyl-amine or potassiumcarbonate stirring is continued at r.t. or elevated temperature. Afterevaporation, the residue was redissolved in ethyl acetate, washed withaqueous acid and base, dried and evaporated. If necessary the productwas purified by trituration or by flash-chromatography or used withoutfurther purification.

Compounds of general formula (V) are commercially available, known orcan be prepared by customary methods starting from known carboxylic acidderivatives. Bisarylureas can be prepared by coupling of an amino phenylacetic acid derivative and a phenylisocyanate.

Step C

The removal of the protecting group PG can be performed either by anacid such as trifluoroacetic acid or an base such as potassium hydroxideor lithium hydroxide, depending on the nature of PG. Reactions arecarried out in aqueous, inert organic solvents such as alcohols e.g.methanol or ethanol, ethers e.g. tetrahydrofurane or dioxane or polaraprotic solvents e.g. dimethylformamide. If necessary, mixtures of theabove solvents may be used.

EXAMPLES

Abbreviations

-   AcOH acetic acid-   Boc tert-butyloxycarbonyl-   DCC dicyclohexylcarbodiimid-   DCM dichloromethane-   DIPEA diisopropylethylamine-   EDCI 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidexHCl-   eq. equivalents-   EtOAc ethyl acetate-   FC flash chromatography-   GC gas chromatography-   HATU 2-(7-aza-3-oxido-1H-1,2,3-benzotriazol-1-yl)-1,    1,3,3-tetramethyluronium hexafluorophosphate-   HOBt N-hydroxybenzotriazole monohydrate-   HPLC high performance liquid chromatography-   ICAM-1 intracellular adhesion molecule 1-   IL1 interleukin 1-   LPS lipopolysaccharide-   MAdCAM-1 mucosal addressin cell adhesion molecule 1-   MeOH methanol-   MeCN acetonitril-   min. minutes-   M.p. melting point-   NF-κB nuclear factor κB-   NMR nuclear magnetic resonance-   n.d. not determined-   PE light petroleum (b.p. 40–60 ° C.)-   r.t. room temperature-   R_(f) TLC: R_(f) value=distance spot traveled/distance solvent front    traveled-   TFA trifluoroacetic acid-   TTF tetrahydrofurane-   TLC thin layer chromatography-   TNF-α tumor necrosis factor α-   t_(R) retention time determined by HPLC-   VCAM-1 vascular cell adhesion molecule 1-   VLA-4 very late antigen 4 (α₄β₁ integrin)    General Remarks

In the examples below, all quantitative data, if not stated otherwise,relate to percentages by weight.

Flash chromatography was carried out on silica gel 60, 40–63μm (E.Merck, Darmstadt, Germany).

Thin layer chromatography was carried out, employing silica gel 60 F₂₅₄coated aluminum sheets (E. Merck, Darmstadt, Germany) with the mobilephase indicated.

Melting points were determined in open capillaries and are notcorrected.

The mass determinations were carried out using the electron sprayionization (ESI) method employing loop injection or split injection viaa HPLC system.

Precursor Synthesis

Example I N-(4-Aminophenyl)-N′-(2-methylphenyl)urea

2-Methylphenylisocyanate (24.6 g, 184.9 mmol) was added dropwise at 0°C. to a solution of 1,4-diamino benzene (20.00 g, 184.9 mmol) in 1000 mLEtOAc. After stirring for 2 h at r.t. the product was collected byfiltration (42.7 g, 177.0 mmol). M.p.>300° C.;

TLC (PE/EtOAc 1/4) R_(f) 0.32; ¹H-NMR (400 MHz, D₆-DMSO) δ 2.10 (s, 3H);4.76 (s, 2H); 6.59 (mc, 2H); 6.89 (mc, 1H); 7.07–7.15 (m, 4H); 7.73 (s,1H); 7.85 (mc, 2H); 8.50 (s, 1H).

Example II tert-Butyl4-({[(2-methylphenyl)amino]carbonyl}amino)benzylcarbamate

2-Methylphenylisocyanate (7.57 g, 59.83 mmol) was added dropwise at 0°C. to a solution of (4-amino-benzyl)-carbamic acid tert-butyl ester(13.30 g, 59.83 mmol, prepared analoguous to: Moloney, Gerard P.;Martin, Graeme R.; Mathews, Neil; Mine, Aynsley; Hobbs, Heather; et al.J. Med Chem. 1999, 42, 2504–2526) in 120 mL DCM. The reaction was heatedunder reflux for 16 h, cooled to r.t. and the precipitated product wascollected by filtration and dried in vacuum (19.20 g, 54.00 mmol). M.p.200–202° C.; TLC (PE/EtOAc 1/1) R_(f) 0.65; ¹H NMR (400 MHz, D₆-DMSO) δ1.39 (s, 9H); 2.24 (s, 3H); 4.06 (d, J=6 Hz, 2H); 6.93 (mc, 1H);7.12–7.17 (m, 4); 7.32 (mc, 1H); 7.40 (mc, 2H); 7.85 (mc, 1H); 7.90 (s,1H); 8.98 (s, 1H).

Example III N-[4-(Aminomethyl)phenyl]-N′-(2-methylphenyl)urea

To a solution of tert-butyl4-({[(2-methylphenyl)amino]carbonyl}amino)benzylcarbamate (2.00 g, 5.63mmol) in CH₂Cl₂ (120 mL) TFA (36 mL) was added at 0° C. and stirred for2 h at r.t. The reaction mixture was evaporated and the product wascollected (2.72 g, TFA salt). M.p. 142–143° C.; TLC (PE/EtOAc 3/2) R_(f)0.14; ¹H NMR (400 MHz, D₆-DMSO) δ 2.24 (s, 3H); 3.97 (q, J=5 Hz, 2H);6.96 (mc, 1H); 7.13–7.19 (m, 2); 7.36 (mc, 2H); 7.51 (mc, 2H); 7.81 (mc,2H); 8.06 (s, 1H); 8.08 (s, 3H); 9.23 (s, 1H).

Compound Synthesis, Example 1

Step A

Example IV 4-{[4-(ethoxycarbonyl)phenyl]amino}-4-oxo-2-phenylbutanoicacid

Ethyl 4-aminobenzoate (910 mg, 5.50 mmol) and phenyl succinic anhydride(1070 mg, 6.00 mmol) were dissolved in 40 mL abs. CH3CN and stirredovernight at r.t. The solid precipitate was isolated and digirated with2-propanol, dried in vacuum, dissolved in DCM and washed with citricacid. 4-{[4-(methoxycarbonyl)-phenyl]amino}-4-oxo-3-phenylbutanoic acidwas isolated as a crystalline solid (980 mg, 2.88 mmol). M.p. 212° C.;TLC (cyclohexane/EtOAc 7/3) R_(f) 0.17; 1H NMR (400 MHz, D₆-DMSO) δ 1.39(t, J=7.2 Hz, 3H); 2.75 (dd, J=3.7 Hz, J=17.2 Hz, 1H); 3.42 (dd, J=3.7Hz, J=17.2 Hz, 1H); 4.09 (dd, J=3.7 Hz, J=17.2 Hz, 1H); 4.35 (q, J=7.2Hz, 2H); 7.33–7.41 (m, 5H), 7.75 (mc, 2H), 7.94 (mc, 2H). Theregiochemistry was determined using NOESY NMR spectroscopy.

Example V 4-{[4-(ethoxycarbonyl)phenyl]amino}-4-oxo-3-phenylbutanoicacid

The solvent was evaporated and the residue was purified by flashchromatography yielding 4-{[4-(methoxycarbonyl)phenyl]amino}-4-oxo-3-phenylbutanoic acid (690 mg,2.87 mmol) M.p. 190° C.; TLC (DCM/MeOH 9/1) R_(f) 0.21; ¹H NMR (400 MHz,D₆-DMSO) δ 1.30 (t, J=7.1 Hz, 3H); 2.23 (s, 3H); 2.80 (mc, 1H); 3.25(mc, 1H); 4.25–4.30 (m, 3H); 3.74 (s, 3H); 6.00 (bs, 1H); 6.65 (d, J=6.6Hz, 2H); 6.90–6.96 (m, 2H); 7.10–7.19 (m, 4H); 7.34–7.38 (m, 2H);7.43–7.51 (m, 2H); 7.69–7.71 (m, 1H); 7.79–7.90 (m, 2H); 8.70 (s, 1H);8.97 (s, 1H).

Step B

Example V Ethyl4-[(4-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]-amino}-4-oxo-3-phenylbutanoyl)amino]benzoate

4-{[4-(Ethoxycarbonyl)phenyl]amino}-4-oxo-2-phenylbutanoic acid (400 mg,1.17 mmol) was dissolved in MeCN (10 mL),1-(3-dimethylaminopropyl)-3-ethyl-carbodiimidhydrochlorid (247 mg, 1.29mmol), 1-hydroxy-1H-benzotriazol (158 mg, 1.17 mmol) followed byN-(4-aminophenyl)-N′-(2-methylphenyl)urea (282 mg, 5 1.17 mmol) wasadded at r.t and the reaction mixture was stirred for 24 h. Ethyl 4-[(4-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-4-oxo-3-phenyl-butanoyl)amino]benzoate (194 mg, 0.34mmol) was isolated by filtration as a colorless solid. M.p.>220° C.; TLC(DCM/MeOH 9/1) R_(f) 0.21; ¹H NMR (400 MHz, D₆-DMSO) δ 1.30 (t, J=7.1Hz, 3H); 2.23 (s, 3H); 2.80 (mc, 1H); 3.25 (mc, 1H); 4.25–4.30 (m, 3H);3.74 (s, 3H); 6.00 (bs, 1H); 6.65 (d, J=6.6 Hz, 2H); 6.90–6.96 (m, 2H);7.10–7.19 (m, 4H); 7.34–7.38 (m, 2H); 7.43–7.51 (m, 2H); 7.69–7.71 (m,1H); 7.79–7.90 (m, 2H); 8.70 (s, 1H); 8.97 (s, 1H).

TABLE 2 The following examples were prepared according to the generalprocedure Ex.- No Structure Name M.p. (° C.) VI

Ethyl4-[(4-{[4-({[2-methyl-phenyl)amino]carbonyl}ami-no)phenyl]amino}-4-oxo-2-phenyl-butanoyl)amino]-ben-zoate152 VII

Ethyl4-[(4-{[4-({[2-methyl-phenyl)amino]carbonyl}ami-no)benzyl]amino}-4-oxo-3-phenyl-butanoyl)amino]ben-zoate132 VIII

Ethyl4-[(4-{[4-({[2-methyl-phenyl)amino]carbonyl}ami-no)benzyl]amino}-4-oxo-2-phenyl-butanoyl)amino]-ben-zoate168Step C

Example 14-[(4-{[4-({[(2-Methylphenyl)amino]carbonyl}amino)phenyl]-amino}-4-oxo-3-phenylbutanoyl)amino]benzoicacid

Ethyl4-[(4-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-4-oxo-3-phenylbutanoyl)amino]benzoate(160 mg, 0.28 mmol) was dissolved in water:THF (10 mL; 1:1; v:v) andLiOH (27 mg, 1.13 mmol) was added at 0° C. and the reaction mixture wasstirred at r.t. for 24 h. The reaction mixture was acidified, theproduct was isolated by filtration and purified by flash chromatography(CH₂Cl₂/MeOH/AcOH; 10/1/0.5) (11 mg, 0.02 mmol). M.p. 169° C. TLC(DCM/MeOH 9/1) R_(f) 0.27, ESI-MS: 537[M+H]⁺.

TABLE 3 The following examples were prepared according to the generalprocedure M.p. No Structure Name (° C.) 2

4-[(4-{[4-({[(2-Methyl-phenyl)amino]carbonyl}-ami-no)phenyl]amino}-4-oxo-2-phenyl-butanoyl)amino]benzoicacid n.d. 3

4-{[(1-(3,4-Dimethoxy-phenyl)-3-{[4-({[(2-methyl-phenyl)amino]carbonyl}ami-no)benzyl]amino}-3-oxo-propyl)amino]carbonyl}benzoicacid 155 4

4-[(4-{[4-({[(2-Methyl-phenyl)amino]carbonyl}-ami-no)benzyl]amino}-4-oxo-2-phenyl-butanoyl)-ami-no]benzoicacid 126In Vitro Assay: Adhesion of Ramos Cells to Immobilized VCAM-1 (Domains1–3)Preparation of VCAM-1 (Extracellular Domains 1–3)

Complementary DNA (cDNA) encoding 7-domain form of VCAM-1 (GenBankaccession #M60335) was obtained using Rapid-Screen™ cDNA library panels(OriGene Technologies, Inc) at Takara Gene Analysis Center (Shiga,Japan). The primers used were 5′-CCA AGG CAG AGT ACG CAA AC-3′ (sense)and 5′-TGG CAG GTA TTA TTA AGG AG-3′ (antisense). PCR amplification ofthe 3-domain VCAM-1 cDNA was perform using Pƒ. DNA polymerase(Stratagene) with the following sets of primers: (U-VCAMdl-3) 5′-CCA TATGGT ACC TGA TCA ATT TAA AAT CGA GAC CAC CCC AGA A-3′; (L-VCAMdl-3)5′-CCA TAT AGC AAT CCT AGG TCC AGG GGA GAT CTC AAC AGT AAA-3′. PCR cyclewas 94° C. for 45 sec, 55° C. for 45 sec, 72° C. for 2 min, repeating 15cycles. After the purification of the PCR product, the fragment wasdigested with KpnI-AvrII. The digested fragment was ligated intopBluescript IISK(−) (Strategene), which was linearized by digesting withKpnI-XhoI. The ligation was followed by transformation to a Dam/Dcmmethylase-free E. coli strain SCS110 (Strategene) to create the donorplasmid pHH7. To direct VCAM-1 molecule into the insect cell secretorypathway, the VCAM-1 coding sequence was fused to signal peptide sequenceof honeybee melittin. The resulting melittin-VCAM fusion was placed incorrect orientation to the baculovirus polyhedrin promoter. Baculovirustransfer vector containing first 3-domain form VCAM-1 (pH10) wasconstructed by ligation of 0.9 kb fragment from AvrII/Klenow/BclIdigests of pH7 into SalI/Klenow/BamHI digests of pMelBacB (Invitrogen).Recombinant baculovirus was generated by using Bac-N-Blue™ Transfectionkit (Invitrogen) according to the manufacture's instruction. Therecombinant virus was amplified by infection to High-Five™ insect cellsfor 5–6 days, and virus titer was determined by plaque assay.

High-Five™ insect cells were pelleted in a 225 ml conical tube bycentrifugation at 1000 rpm for 5 min. After discarding the supernatant,the pellet was resuspended in 1.5×10⁹ pfu (MOI=5) of high-titer virussolution, followed by incubation for 1.5 hours at room temperature. Thecells were pelleted again and washed once in fresh Express Five™ serumfree medium. The cells were pelleted again and finally, resuspended in200 ml of fresh Express Five TM medium, transferred to a 1,000 ml shakerflask, and incubated in a shaker at 27° C., 130 rpm, for 48 hours beforethe culture supernatant was collected. The purification of 3-domain formof VCAM-1 from the culture supernatant was performed by one-step anionexchange chromatography. Protein concentration was determined by usingCoomassie protein assay reagent (Pierce) according to the manufacture'sinstruction.

Preparation of VCAM-1 Coated Microtiter Plates

Recombinant human VCAM-1 (extracellular domains 1–3) was dissolved at1.0 μg/ml in PBS. Each well of the microtiter plates (Nalge NuncInternational, Fluoronunc Cert, 437958) was coated with 100 μl ofsubstrate or for background control with buffer alone for 15 hours at 4°C. After discarding the substrate solution, the wells were blocked using150 μl per well of block solution (Kirkegaard Perry Laboratories,50-61-01) for 90 minutes. The plate was washed with wash buffercontaining 24 mM Tris-HCl (pH 7.4), 137 mM NaCl, 27 mM KCl and 2 mMMnCl₂ just before addition of the assay.

In Vitro Assay Using Ramos Cells

Preparation of Fluorescence Labeled Ramos Cells

Ramos cells (American Type Culture Collection, Clone CRL-1596) werecultured in RPMI 1640 medium (Nikken Bio Medical Laboratory, CM1101)supplemented with 10% fetal bovine serum (Hyclone, A-1119-L), 100 U/mlpenicilin (Gibco BRL, 15140–122) and 100 μg/ml streptomycin (Gibco BRL,15140–122) in a humidified incubator at 37° C. with 5% CO₂.

Ramos cells were incubated with phosphate balanced solution (PBS,Nissui, 05913) containing 25 μM of 5(-and 6)-carboxyfluoresceindiacetate, succinimidyle ester (CFSE, Dojindo Laboratories, 345–06441)for 20 min at room temperature while gently swirling every 5 min. Aftercentrifugation at 1000 rpm for 5 min, the cell pellet was resuspendedwith adhesion assay buffer at a cell density of 4×10⁶ cells/ml. Theadhesion assay buffer was composed of 24 mM Tris-HCl (pH 7.4), 137 mMNaCl, 27 mM KCl, 4 mM glucose, 0.1 % bovine serum albumin (BSA, Sigma,A9647) and 2 mM MnCl₂.

Assay Procedure (Ramos Cells)

The assay solution containing each test compounds or 5 μg/ml anti-CD49dmonoclonal antibody (Immunotech, 0764) was transferred to the VCAM-1coated plates. The final concentration of each test compounds was 5 μM,10 μM or various concentrations ranging from 0.0001 μM to 10 μM using astandard 5-point serial dilution. The assay solution containing thelabeled Ramos cells was transferred to the VCAM-1 coated plates at acell density of 2×10⁵ cells per well and incubated for 1 hour at 37° C.The non-adherent cells were removed by washing the plates 3 times withwash buffer. The adherent cells were broken by addition of 1 % TritonX-100 (Nacalai Tesque, 355-01). Released CFSC was quantifiedfluorescence measurement in a fluorometer (Wallac, ARVO 1420 multilabelcounter).

The adhesion of Ramos cells to VCAM-1 was analyzed by percent bindingcalculated by the formula:

100×(FTS−FBG)/(FTB−FBG)=% binding, where FTB is the total fluorescentintensity from VCAM-1 coated wells without test compound; FBG is thefluorescent intensity from wells with anti-CD49d monoclonal antibody andFTS is the fluorescent intensity from wells containing the test compoundof this invention.In Vitro Activity

In the Ramos VCAM-1 assay the observed IC₅₀ value ranges are indicatedin Table 4.C>10 μM≧B>1 μM≧A

TABLE 4 No IC₅₀ 1 A 2 C 3 C 4 A

1. A compound of the general formula (I)

wherein R¹ represents a 4- to 9-membered saturated, unsaturated oraromatic cyclic residue, which can contain 0 to 3 heteroatoms selectedindependently from the group N, S and O, and wherein R¹is substituted by—R¹⁻¹—Z, wherein R¹⁻¹ represents a bond, —O—,—S—, NR¹⁻², C₁–C₁₀ alkenyl,C₂–C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-memberedsaturated or unsaturated heterocyclic residue containing up to 3heteroatoms selected from the group oxygen, nitrogen and sulfur, whereinR¹⁻¹can optionally be substituted by 1 to 2 substitutes selected fromthe group R¹⁻³, wherein R¹⁻²can optionally be hydrogen, C₁–C₁₀ alkyl,C₂–C₁₀ alkenyl or C₂–C₁₀ alkynyl, and wherein R¹⁻³represents hydrogen,C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀alkynyl, C₆ or C₁₀ aryl, C₃–C₇cycloalkyl or a 4–9-membered saturated or unsaturated heterocyclicresidue containing up to 3 heteroatoms selected from the group oxygen,nitrogen and sulfur, represents —C(O)OR^(Z-1), —C(O)NR^(Z-2)R^(Z-3, —SO)₂NR^(Z-2)R^(Z-3), —SO(OR^(Z-1)), —SO₂(OR_(Z-1)), —P(O)R^(Z-1)(OR^(Z-3))or —PO(OR^(Z-1))(OR^(Z-3),) wherein R^(z-2) is hydrogen, C₁–C₄ alkyl,C₂–C₆ alkenyl, C₂–C₆ alkynyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,—C(O)R^(z-4) or —SO₂R^(z-4), wherein R^(z-4) is C₁–C₄ alkyl, C₂–C₆alkenyl, C₂–C₆ alkynyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl, R^(z-1) andR^(z-3) are independently selected from the group hydrogen, C₁–C₄ alkyl,C₂ –C₆ alkenyl, C₂ –C₆ alkynyl, C₃ –C₆ cycloalkyl, C₆ or C₁₀ aryl andbenzyl, wherein R^(Z-1) and R^(Z-3) can optionally be substituted by 1to 3 substitutes selected from the group C₁–C₄ alkyl, C₁–C₄ alkyloxy,halogen, nitro, and cyano, and wherein R¹ can optionally be substitutedby 0 to 2 substitutes selected from R¹⁻⁴, halogen, nitro, amino, cyanoand oxo, wherein R¹⁻⁴ is selected from the group C₁–C₄ alkyl, C₁–C₄alkyloxy, phenyl, phenoxy, phenylamino and C₃–C₆ cycloalkyl, R²represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁o alkynyl, C₆ orC₁₀ aryl or C₃–C₇ cycloalkyl, wherein R² can optionally be substitutedby 1 to 3 radicals independently selected from the group C₁C₄ alkyl,trifluormethyl, trifluormethoxy, halogen, cyano, nitro and oxo, R³represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀ alkynyl, C₆ orC₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered saturated or unsaturatedheterocyclic residue containing up to 2 heteroatoms selected from thegroup oxygen, nitrogen and sulfur, wherein R³ can optionally besubstituted by 1 to 3 radicals R³⁻¹, wherein R³ represents C₁–C₄ alkyl,trifluormethyl, trifluormethoxy, —OR³⁻², —SR³⁻², NR³⁻³R³⁻⁴, —C(O)R³⁻²,S(O)R³⁻², —S)₂R³⁻², —OC(O)R³⁻², —C(O)NR³⁻³R³⁻⁴, —NR³⁻²C(O)R³⁻³,—SO₂NR³⁻³R³⁻⁴, NR³⁻²SO₂R³⁻³, —NR³⁻²C(O)NR³⁻³R³⁻⁴, —NR³⁻²C(O)OR³⁻³,—OC(O)NR³⁻³R³⁻⁴, —CO₂R³⁻⁵, halogen, cyano, nitro or oxo, wherein R³⁻²represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,wherein R³⁻³ and R³⁻⁴ are independently selected from the grouphydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl and benzyl, andwherein R³⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,R⁴ represents hydrogen, C₁–C₁₀alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀ alkynyl, C₆or C₁₀ aryl, C₃–C₇ cycloalkyl or a 4–9-membered saturated or unsaturatedheterocyclic residue containing up to 2 heteroatoms selected from thegroup oxygen, nitrogen and sulfur, wherein R⁴ can optionally besubstituted by 1 to 3 radicals R⁴⁻¹, wherein R⁴represents C_(1–C) ₄alkyl, trifluormethyl, trifluormethoxy, —OR⁴⁻², —SR⁴⁻², NR⁴⁻³ R⁴⁻¹,—C(O)R⁴⁻², S(O)R⁴⁻², —S₂R⁴⁻², —OC(O)R⁴⁻², —C(O)NR⁴R⁴⁻⁴, —NR⁴⁻²C(O)R⁴⁻³,—S₂NR⁴⁻³R⁴⁻⁴, NR⁴⁻²S₂R⁴⁻³, —NR⁴⁻²C(O)NR⁴⁻³R⁴⁻⁴, —NR⁴⁻²C(O)OR⁴⁻³,—OC(O)NR⁴⁻³R⁴⁻⁴, —C₂R⁴⁻⁵, halogen, cyano, nitro or oxo, wherein R⁴⁻²represents hydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl,wherein R⁴⁻³ and R⁴⁻⁴ are independently selected from the grouphydrogen, C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl and benzyl, andwherein R⁴⁻⁵ represents C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl orR³ and R⁴ together with the carbon atom to which they are attached forma 4–7-membered saturated or unsaturated ring containing up to 2heteroatoms selected from the group oxygen, nitrogen and sulfur, whichcan optionally be substituted by 1 to 2 substitutes selected from thegroup C₁–C₄ alkyl, phenyl, benzyl, C₃–C₇, cycloalkyl, C₁–C₄ alkyloxy,halogen, nitro, cyano and oxo and which can be fused with a 3–7 memberedhomocyclic or heterocyclic, saturated, unsaturated or aromatic ring, R⁵represents hydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀ alkynyl, C₆ orC₁₀ aryl or C₃–C₇, cycloalkyl, wherein R⁵ can optionally be substitutedup to threefold by C₁–C₄ alkyl, trifluormethyl, trifluormethoxy,halogen, cyano, nitro or oxo, R⁶ represents phenyl or a 5- to 6-memberedaromatic heterocyclic residue containing up to 3 heteroatomsindependently selected from the group oxygen, nitrogen and sulfur,wherein R⁶ is substituted by —NR⁶⁻²C(O)NR⁶⁻³R or —NR⁶⁻²C(S)NR⁶⁻³R⁶⁻⁴ andcan furthermore optionally be substituted by halogen, wherein R⁶⁻² andR⁶⁻³ are independently selected from the group hydrogen and C_(1–C) ₄alkyl, or together form a group

and wherein R⁶⁻⁴ represents phenyl, wherein R⁶⁻⁴ can optionally besubstituted by 1–2 substitutes selected from the group C₁–C₄ alkyl,C₁–C₄ alkyloxy, halogen, nitro, trifluormethyl, trifluormethoxy andcyano, or R⁶ represents a group

wherein R⁶⁻¹ represents a substituent selected from the group hydrogen,C₁–C₄ alkyl, C₁–C₄ alkyloxy, halogen, nitro, trifluormethyl,trifluormethoxy and cyano, and wherein R⁶⁻⁵ represents a substituentselected from the group hydrogen, C₁–C₄ alkyl, C₁–C₄ alkyloxy, halogen,nitro, trifluormethyl, trifluormethoxy and cyano, R⁷ representshydrogen, C₁–C₁₀ alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀ alkynyl, C₆ or C₁₀ aryl,C₃–C₇ cycloalkyl or a 4–9-membered saturated or unsaturated heterocyclicresidue containing up to 2 heteroatoms selected from the group oxygen,nitrogen and sulfur, wherein R⁷ can optionally be substituted by 1 to 3radicals R⁷⁻¹, wherein R⁷⁻¹ represents C₁–C₄ alkyl, trifluormethyl,trifluormethoxy, —OR⁷⁻², —SR⁷⁻², NR⁷⁻³R⁷⁻⁴, —C(O)R⁷⁻², S(O)R⁷⁻²,—S₂R⁷⁻², —OC(O)R⁷⁻², —C(O)NR⁷⁻³R⁷⁻⁴, —NR⁷⁻²C(O)R⁷⁻³, —S₂NR⁷⁻³R⁷⁻⁴,NR⁷⁻²S₂R⁷⁻³, —NR⁷⁻²C(O)NR⁷⁻³R⁷⁻⁴, —NR⁷⁻²C(O)OR⁷⁻³, —OC(O)NR⁷⁻³ R⁷⁻⁴,—C₂R⁷⁻⁵, halogen, cyano, nitro or oxo, wherein R⁷⁻² represents hydrogen,C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl, wherein R⁷⁻³ and R⁷⁻⁴ areindependently selected from the group hydrogen, C₁–C₄ alkyl, C₃–C₆cycloalkyl, C₆ or C₁₀ aryl and benzyl, and wherein R⁷⁻⁵ represents C₁–C₄alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl R⁸ represents hydrogen, C₁–C₁₀alkyl, C₂–C₁₀ alkenyl, C₂–C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃–C₇ cycloalkylor a 4–9-membered saturated or unsaturated heterocyclic residuecontaining up to 2 heteroatoms selected from the group oxygen, nitrogenand sulfur, wherein R⁸ can optionally be substituted by 1 to 3 radicalsR⁸⁻¹, wherein R⁸⁻¹ represents C₁–C₄ alkyl, trifluormethyl,trifluormethoxy, —OR⁸⁻², —SR⁸⁻², NR⁸⁻³R⁸⁻⁴, —C(O)R⁸⁻², S(O)R⁸⁻²,—SO₂R⁸⁻², —OC(O)R⁸⁻², —C(O)NR⁸⁻³R⁸⁻⁴, —NR⁸⁻²C(O)R⁸⁻³, —SO₂NR⁸⁻³R⁸⁻⁴,NR⁸⁻²SO₂R⁸⁻³, —NR⁸⁻²C(O)NR⁸⁻³R⁸⁻⁴, —NR⁸⁻²C(O)OR⁸⁻³, —OC(O)NR⁸⁻³R⁸⁻⁴,—CO₂R⁸⁵, halogen, cyano, nitro or oxo, wherein R⁸⁻² represents hydrogen,C₁–C₄ alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl, wherein R⁸⁻³ and R⁸⁻⁴ areindependently selected from the group hydrogen, C₁–C₄ alkyl, C₃–C₆cycloalkyl, C₆ or C₁₀ aryl and benzyl, and wherein R⁸⁻⁵ represents C₁–C₄alkyl, C₃–C₆ cycloalkyl, C₆ or C₁₀ aryl or R⁷ and R⁸ together form a4–7-membered saturated or unsaturated ring containing up to 2heteroatoms selected from the group oxygen, nitrogen and sulfur, whichcan optionally be substituted by 1 to 2 substitutes selected from thegroup C₁–C₄ alkyl, phenyl, benzyl, C₃–C₇ cycloalkyl, C₁–C₄ alkyloxy,halogen, nitro, cyano, and oxo and which can be fused with a 3–7membered homocyclic or heterocyclic, saturated, unsaturated or aromaticring, X represents a bond or (—CR^(Z-1)R^(X-2)—)_(n), wherein R^(X-1)and R^(X-2) can be independently selected from the group hydrogen, C₁–C₄alkyl, C₂–C₄ alkenyl, and C₂–C₄ alkynyl, wherein R^(X-1) and R^(X-2) canoptionally independently be substituted by 1 to 2 substitutes selectedfrom the group C₁–C₄ alkyl, phenyl, benzyl, C₃–C₇ cycloalkyl, C₁–C₄alkyloxy, halogen, nitro, cyano, and oxo, and wherein n is an integer 0or 1, or a pharmaceutically acceptable salt thereof.
 2. The compound ofgeneral formula (I) according to claim 1, wherein R¹ represents a phenylring.
 3. The compound according to claim 1 or 2, wherein R¹⁻¹ representsa bond and Z represents COOR^(Z-1), wherein R^(Z-1) has the meaningindicated in claim
 1. 4. The compound according to claim 1, wherein R⁶represents phenyl, which is substituted by —NHC(O)NHR⁶⁻⁴, wherein R⁶⁻⁴is substituted with methyl or trifluormethoxy.
 5. The compound accordingto claim 1, wherein X represents a methylene group.
 6. The compoundaccording to claim 1, wherein one of the substitutes R³, R⁴, R⁷ and R⁸represents phenyl, which can optionally be substituted by up to threesubstitutes independently selected from the group C₁–C₄-alkyl andhalogen, and the remaining substitutes represent hydrogen.
 7. A processfor preparation of compounds of general formula (I) according to claim1, which comprises reacting a carboxylic acid of general formula (I′)

or activated derivative thereof, wherein R¹, R², R³, R⁴, R⁷ and R⁸ havethe meaning shown in claim 1, with a compound of the general formula(I″)R⁶—X—NR⁵H  (I″) wherein X, R⁵ and R⁶ have the meaning shown in claim 1,in inert solvent.
 8. A method for the treatment or the prevention of acondition mediated by integrins, comprising administering an effectiveamount of a compound of claim
 1. 9. A method for the treatment or theprevention of atherosclerosis, asthma, chronic obstructive pulmonarydisease (COPD), allergies, diabetes, inflammatory bowel disease,multiple sclerosis, myocardial ischemia, rheumatoid arthritis,transplant rejection and other inflammatory, autoimmune and immunedisorders, comprising administering an effective amount of a compound ofclaim
 1. 10. A pharmaceutical composition, comprising a compoundaccording to claim 1 and a pharmaceutically acceptable carrier.
 11. Acompound of general formula (I) according to claim 2, characterized inthat R¹ is a 1,4-substituted phenyl ring.